Triggering sleep slow waves by transcranial magnetic stimulation

During much of sleep, cortical neurons undergo near-synchronous slow oscillation cycles in membrane potential, which give rise to the largest spontaneous waves observed in the normal electroencephalogram (EEG). Slow oscillations underlie characteristic features of the sleep EEG, such as slow waves and spindles. Here we show that, in sleeping subjects, slow waves and spindles can be triggered noninvasively and reliably by transcranial magnetic stimulation (TMS). With appropriate stimulation parameters, each TMS pulse at <1 Hz evokes an individual, high-amplitude slow wave that originates under the coil and spreads over the cortex. TMS triggering of slow waves reveals intrinsic bistability in thalamocortical networks during non-rapid eye movement sleep. Moreover, evoked slow waves lead to a deepening of sleep and to an increase in EEG slow-wave activity (0.5-4.5 Hz), which is thought to play a role in brain restoration and memory consolidation.     

A physical approach to modify the hydraulic reactivity of α-tricalcium phosphate powder

A microsized α-tricalcium phosphate (α-TCP) powder was calcined at various temperatures (350 °C < T < 800 °C) for various durations (1–24 h) and the resulting physico-chemical and reactivity changes were measured. Without calcination, the α-TCP powder started reacting within minutes after contacting a 0.2 M Na₂HPO₄ solution as measured by isothermal calorimetry. The overall reaction was finished within a few days. After calcination at 350 °C  T  550 °C for 24 h, no significant changes in the crystalline composition, crystallite size, particle size or specific surface area were noticed. However, the powder reactivity was progressively changed. More specifically, the hydraulic reaction of the powders calcined at 500 and 550 °C only started after 2–3 h whereas the overall hydraulic reaction was only slightly postponed, suggesting that physical or chemical changes had occurred at the particle surface. As mainly physical changes were detected at the particle surface during calcination at 500 °C, it was speculated that the appearance of this reaction delay (= induction time) was due to the disappearance of surface defects during the calcination step, i.e. to the need to create surface defects to induce dissolution and hence reaction.     

Role of CD11b+ Macrophages in Intraperitoneal Lipopolysaccharide-Induced Aberrant Lymphangiogenesis and Lymphatic Function in the Diaphragm

Lymphatic vessels in the diaphragm are essential for draining peritoneal fluid, but little is known about their pathological changes during inflammation. Here we characterized diaphragmatic lymphatic vessels in a peritonitis model generated by daily i.p. administration of lipopolysaccharide (LPS) in mice. Intraperitoneal LPS increased lymphatic density, branching, sprouts, connections, and network formation in the diaphragm in time- and dose-dependent manners. These changes were reversible on discontinuation of LPS administration. The LPS-induced lymphatic density and remodeling occur mainly through proliferation of lymphatic endothelial cells. CD11b⁺ macrophages were massively accumulated and closely associated with the lymphatic vessels changed by i.p. LPS. Both RT-PCR assays and experiments with vascular endothelial growth factor-C/D blockade and macrophage-depletion indicated that the CD11b⁺ macrophage-derived lymphangiogenic factors vascular endothelial growth factor-C/D could be major mediators of LPS-induced lymphangiogenesis and lymphatic remodeling through paracrine activity. Functional assays with India ink and fluorescein isothiocyanate-microspheres indicated that impaired peritoneal fluid drainage in diaphragm of LPS-induced peritonitis mice was due to inflammatory fibrosis and massive attachment of CD11b⁺ macrophages on the peritoneal side of the diaphragmatic lymphatic vessels. These findings reveal that CD11b⁺ macrophages play an important role in i.p. LPS-induced aberrant lymphangiogenesis and lymphatic dysfunction in the diaphragm.The peritoneum provides the lining of the peritoneal cavity and is the most extensive serous membrane in the body.¹ The peritoneal membrane is formed by a single layer of mesothelial cells. Beneath the mesothelial cells, there is a very thin and discontinuous layer of connective tissue and a layer of fenestrated lymphatic vessels.² These three layers not only function as an absorptive surface for peritoneal fluid but also remove pathogens and prevent cells from leaking through damage in the gastrointestinal tract or ascending through the female genital tract.^3,4 The peritoneum also plays crucial roles in the local defensive response against bacterial invasion with appropriate activation of resident immune cells and the recruitment of circulating immune cells.^5,6Lymphatic vessels have distinctive morphologies and functions in different tissues and organs.^7,8 Lymphatic vessels play an essential role in the maintenance of tissue fluid homeostasis through regulated uptake of protein-rich interstitial fluid into draining lymphatic vessels and transport of the drained lymphatic fluid into the blood vasculature via collecting lymphatic vessels.⁹ In addition, lymphatic vessels have roles in lipid absorption, antigen presentation, tumor metastasis, and wound healing.^{10,11,12,13,14,15,16} Lymphatic vessels beneath the peritoneum, particularly lymphatic vessels on the peritoneal side of the muscular region of diaphragm, provide the central route for draining peritoneal fluid.^2,17,18Lymphatic vessels on the peritoneal side of the diaphragm are largely attenuated but well designed for fluid absorption with extremely flattened and broad lumina (also called lacunae), which are connected with openings between mesothelial cells covering the peritoneal surface.^2,19 In comparison, lymphatic vessels on the pleural side of diaphragm are tubular, like other lymphatic vessels.^20,21 There are seven to nine parallel lymphatic strips on each hemisphere (sterno-costal muscular region) of the peritoneal side of diaphragm, and these lymphatic vessels are directly connected to the tubular lymphatic vessels on the pleural side by transmural lymphatic branches.^20,21,22 Therefore, the peritoneal fluid absorbed by lymphatic lacunae is directly transported into the lymphatic vessels on the pleural side. In contrast, there are few lymphatic vessels in the central tendon region of the diaphragm. However, little is known about relationship between the structural and functional changes of diaphragmatic lymphatic vessels and peritoneal illnesses.Our understanding of the molecular and cellular regulation of new lymphatic vessel formation, “lymphangiogenesis,” has greatly advanced in recent years.¹⁰ Among lymphangiogenic growth factors, the roles of vascular endothelial growth factor (VEGF)-C and VEGF-D (VEGF-C/D) and their lymphatic vessel-specific receptor VEGF receptor-3 (VEGFR-3) are specific and essential in lymphangiogenesis.¹⁰ In addition, VEGF-A and its receptors play additional roles in lymphangiogenesis in certain pathological conditions.^10,11 Moreover, proinflammatory cytokine-induced activation of macrophages is closely involved in pathological lymphangiogenesis in tracheal mucosa and cornea by reciprocal interactions with the VEGF-C/D-VEGFR-3 system.^12,13,14,15 However, the relationship between proinflammatory cytokine-induced activation of macrophages and pathological changes of diaphragmatic lymphatic vessels during peritonitis is unknown.In this study, we investigated how acute inflammatory peritonitis affects the diaphragmatic lymphatic vessels structurally and functionally and what roles are played by activated macrophages in this situation. To generate a peritonitis model, we administered lipopolysaccharide (LPS; endotoxin) directly into the peritoneal cavity of mouse. LPS is a well-known cell wall component of most Gram-negative bacteria and acts as a potent initiator of inflammation.^23,24 Interestingly, mice with LPS-induced peritonitis displayed aberrant lymphangiogenesis, lymphatic remodeling, and lymphatic dysfunction in the diaphragm. We have defined the underlying mechanisms and the responsible molecules by using specific blocking agents to reveal the roles of critical effector cells and molecules. Our results show that CD11b⁺ macrophages have an important role in LPS-induced aberrant lymphangiogenesis and lymphatic dysfunction in the diaphragm.     

A novel type of interplexiform amacrine cell in the mouse retina

Mammalian retinas comprise an enormous variety of amacrine cells with distinct properties and functions. The present paper describes a new interplexiform amacrine cell type in the mouse retina. A transgenic mouse mutant was used that expressed the gene for the enhanced green fluorescent protein (EGFP) instead of the coding DNA of connexin45 in several retinal cell classes, among which a single amacrine cell population was most prominently labelled. Staining for EGFP and different marker proteins showed that these amacrine cells are interplexiform: they stratify in stratum S4/5 of the inner plexiform layer and send processes to the outer plexiform layer. These cells were termed IPA-S4/5 cells. They belong to the group of medium-field amacrine cells and are coupled homologously and heterologously to other amacrine cells by connexin45. Immunostaining revealed that IPA-S4/5 cells are GABAergic and express GAT-1, a plasma-membrane-bound GABA transporter possibly involved in non-vesicular GABA release. To characterize the light responses of IPA-S4/5 cells, patch-clamp recordings in retinal slices were made. Consistent with their stratification in the ON sublamina of the inner plexiform layer, cells depolarized in response to light ON stimuli and transiently hyperpolarized in response to light OFF. Responses of cells to green (578 nm) and blue (400 nm) light suggest that they receive input from cone bipolar cells contacting both M- and S-cones, possibly with reduced S-cone input. A new type of interplexiform ON amacrine cell is described, which is strongly coupled and uses GABA but not dopamine as its neurotransmitter.     

Encecalol angelate, an unstable chromene from Ageratum conyzoides L.: total synthesis and investigation of its antiprotozoal activity

Ethnopharmacological relevance

In agreement with ethnomedicinal reports, the dichloromethane extract of Ageratum conyzoides L. (Asteraceae) was recently shown to be of considerable activity against Trypanosoma brucei rhodesiense, the etiologic agent of East African Human Trypanosomiasis (East African Sleeping Sickness). Isolated compounds, namely, methoxylated flavonoids as well as the chromene derivative encecalol methyl ether, were less active than the crude extract. The activity of the extract was found to decrease considerably while stored in solution. An unstable compound was detected in the fresh extract by HPLC, which was converted rapidly into the encecalol methyl ether while stored in methanolic solution. This compound, deemed to represent a constituent with antitrypanosomal activity, could not be isolated from the extract in intact form.

Aim of the study

To elucidate the structure of this unstable compound and to investigate its potential role in the antitrypanosomal activity of the total extract.

Materials and Methods

UHPLC/ESI-qQTOF MSMS and NMR data of the degraded product indicated its chemical identity as encecalol angelate (1) which was therefore prepared by total synthesis via a linear six steps synthesis, starting from resorcinol and 2-methylbut-3-en-2-ol.

Results

Total synthesis, in an overall yield of 15%, led to pure 1, which was chromatographically and spectroscopically identical with the natural product. The compound degraded in methanol with a half-life of approximately 6 h to yield encecalol methyl ether (2). The antiprotozoal activity of synthetic encecalol angelate against T. brucei rhodesiense as well as T. cruzi, Leishmania donovani and Plasmodium falciparum was investigated and found to be quite low.

Conclusions

The synthetic approach applied here for the first time also provides access to the related bioactive chromenes encecalin (7) and encecalol (8) with improved yields compared with reported methods. Encecalol angelate, however, is most likely not responsible for the high antitrypanosomal activity of the freshly prepared dichloromethane extract of A. conyzoides.     

Antiprotozoal activity of Melampyrum arvense and its metabolites

An activity guided isolation of the H₂O subextract of the crude extract of Melampyrum arvense L. afforded iridoid glucosides: aucubin (1), melampyroside (2), mussaenoside (3), mussaenosidic acid (4), 8‐epi‐loganin (5); flavonoids: apigenin (6), luteolin (7), luteolin 7‐O‐β‐glucopyranoside (8); a lignan glycoside dehydrodiconiferyl alcohol 9‐O‐β‐glucopyranoside (9); and benzoic acid (10). β‐Sitosterol (11) and a fatty acid mixture (12) were identified as the active principles of the CHCl₃ subextract. The structures of the isolates were elucidated by spectroscopic methods, while the composition of 12 was identified by GC‐MS after methylation. Luteolin (7) appeared as the most active compound against Trypanosoma brucei rhodesiense and Leishmania donovani (IC₅₀ values 3.8 and 3.0 μg/mL). Luteolin 7‐O‐β‐glucopyranoside (8) displayed the best antiplasmodial activity against Plasmodium falciparum (IC₅₀ value 2.9 μg/mL). This is the first detailed phytochemical study on Turkish M. arvense and the first report of the antiprotozoal effect of Melampyrum species and its constituents. Copyright © 2010 John Wiley & Sons, Ltd.     

Soluble vascular endothelial growth factor receptor-3 suppresses lymphangiogenesis and lymphatic metastasis in bladder cancer

Background  

Most bladder cancer patients experience lymphatic metastasis in the course of disease progression, yet the relationship between lymphangiogenesis and lymphatic metastasis is not well known. The aim of this study is to elucidate underlying mechanisms of how expanded lymphatic vessels and tumor microenvironment interacts each other and to find effective therapeutic options to inhibit lymphatic metastasis.